Long-term Preconditioning of Plantlets: a Practical Method for Enhancing Survival of Pineapple (Ananas Comosus (l.) Merr.) Shoot Tips Cryopreserved Using Vitrification

BACKGROUND: The purpose of this study was to develop an efficient cryopreservation protocol for pineapple (Ananas comosus (L.) Merr.) shoot tips. OBJECTIVE: The optimal state of pineapple plantlets was investigated by using sucrose preconditioning to enhance survival after cryostorage. METHODS: To achieve a suitable state of plantlets before cryopreservation, 0.2 M to 0.4 M sucrose concentrations combined with short- (0–7 days), medium- (15–30 days), and long-term (75–150 days) preconditioning periods were compared. RESULTS AND CONCLUSION: The highest survival (100%) was achieved using the following procedure: intact plantlets underwent long-term preconditioning with 0.2 M sucrose for 135 days, dissected shoot tips were treated with a loading solution containing 2.0 M glycerol + 0.4 M sucrose for 60 min at 25°C and the shoot tips were dehydrated in PVS2 for 2 h at 0°C before being plunged in liquid nitrogen. Rewarming was conducted in a water-bath for 30 s at 40°C and PVS2 was replaced with a 1.2 M sucrose solution for 30 min at 25°C. The shoot tips were transferred on semisolid medium and left in the dark for 1 week, then in dim light for 3 weeks.